Limits...
The effect of RNA base lesions on mRNA translation.

Calabretta A, Küpfer PA, Leumann CJ - Nucleic Acids Res. (2015)

Bottom Line: The biological effect of oxidatively damaged RNA, unlike oxidatively damaged DNA, has rarely been investigated, although it poses a threat to any living cell.Alternatively, the unlabeled mRNA construct was used and incubated with (35)S-methionine to prove peptide elongation of the message.We find that all base-lesions interfere substantially with ribosomal translation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.

Show MeSH

Related in: MedlinePlus

(A) Synthesis of the puromycin mRNA constructs via splint ligation of synthetic oligonucleotides. X is the position where the lesions were introduced and Y indicates the encoded amino acids, 9 = triethylene glycol linker, P = puromycin. (B) Translation assay: the hybrid template is labeled with a radioactive phosphate and used in a standard rabbit reticulocyte translation assay. The results are analyzed by PAGE.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482091&req=5

Figure 2: (A) Synthesis of the puromycin mRNA constructs via splint ligation of synthetic oligonucleotides. X is the position where the lesions were introduced and Y indicates the encoded amino acids, 9 = triethylene glycol linker, P = puromycin. (B) Translation assay: the hybrid template is labeled with a radioactive phosphate and used in a standard rabbit reticulocyte translation assay. The results are analyzed by PAGE.

Mentions: To determine the effect of an mRNA lesion on protein biosynthesis we decided to develop an in vitro translation assay (Figure 2) based on the mRNA-display method (26,29,31,32). In this method the encoded peptide becomes covalently linked to the message at the end of translation by a puromycin unit (P) that is attached via a DNA-linker to the 3′-end of the message. Puromycin is a nucleoside that mimics the aminoacyl end of tRNA and covalently binds the nascent peptide by entering the P-site of the ribosome (26). We reasoned that this method is superior to standard translation assays without covalent attachment of the peptide in view of expected difficulties in the isolation of the small peptide out of the translation mixture. Furthermore, the nucleic acid part in the translation product enables easy analysis by polyacrylamide gel electrophoresis (PAGE) from which information on full length, partial length or absence of an attached peptide should be visible by differential gel shifts. 32P-labeling of the mRNA construct before translation can be used to visualize the reaction products. Alternatively, translation with unlabeled mRNA in the presence 35S-methionine can be used to unambiguously prove the covalent inclusion of the peptide.


The effect of RNA base lesions on mRNA translation.

Calabretta A, Küpfer PA, Leumann CJ - Nucleic Acids Res. (2015)

(A) Synthesis of the puromycin mRNA constructs via splint ligation of synthetic oligonucleotides. X is the position where the lesions were introduced and Y indicates the encoded amino acids, 9 = triethylene glycol linker, P = puromycin. (B) Translation assay: the hybrid template is labeled with a radioactive phosphate and used in a standard rabbit reticulocyte translation assay. The results are analyzed by PAGE.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482091&req=5

Figure 2: (A) Synthesis of the puromycin mRNA constructs via splint ligation of synthetic oligonucleotides. X is the position where the lesions were introduced and Y indicates the encoded amino acids, 9 = triethylene glycol linker, P = puromycin. (B) Translation assay: the hybrid template is labeled with a radioactive phosphate and used in a standard rabbit reticulocyte translation assay. The results are analyzed by PAGE.
Mentions: To determine the effect of an mRNA lesion on protein biosynthesis we decided to develop an in vitro translation assay (Figure 2) based on the mRNA-display method (26,29,31,32). In this method the encoded peptide becomes covalently linked to the message at the end of translation by a puromycin unit (P) that is attached via a DNA-linker to the 3′-end of the message. Puromycin is a nucleoside that mimics the aminoacyl end of tRNA and covalently binds the nascent peptide by entering the P-site of the ribosome (26). We reasoned that this method is superior to standard translation assays without covalent attachment of the peptide in view of expected difficulties in the isolation of the small peptide out of the translation mixture. Furthermore, the nucleic acid part in the translation product enables easy analysis by polyacrylamide gel electrophoresis (PAGE) from which information on full length, partial length or absence of an attached peptide should be visible by differential gel shifts. 32P-labeling of the mRNA construct before translation can be used to visualize the reaction products. Alternatively, translation with unlabeled mRNA in the presence 35S-methionine can be used to unambiguously prove the covalent inclusion of the peptide.

Bottom Line: The biological effect of oxidatively damaged RNA, unlike oxidatively damaged DNA, has rarely been investigated, although it poses a threat to any living cell.Alternatively, the unlabeled mRNA construct was used and incubated with (35)S-methionine to prove peptide elongation of the message.We find that all base-lesions interfere substantially with ribosomal translation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.

Show MeSH
Related in: MedlinePlus